*Organism (including strain):Leishmania major strain Friedlin
Etiologic Risk Group (see link below): Risk group 2 (RG2) - parasitic agents
*Background/Disease Information (sort of like the Intro to your Mini Research Write up): Leishmania major is a protozoan species associated with the disease zoonotic cutaneous leishmaniasis (the most common form of leishmaniasis). The intracellular pathogen affects hosts' immune systems by infecting dendritic cells and macrophages. L. major is largely endemic to the Eastern hemisphere, specifically the Middle East, and the northern regions of Africa, China, and India. However, leishmaniasis is also found in the Americas, and has even recently spread to North Texas. There are approximately 1-1.5 million worldwide cases of cutaneous leishmaniasis a year. The disease is transmitted by the bite of female sandflies (spread by vectors). Clinical presentations of cutaneous leishmaniasis include acute infection and lesions at the bite site. Nodules that ulcerate may form. Sores may remain for a few months to a year. The patient's immune system is usually naturally restored within 3-6 months after infection. A current antiprotozoal oral drug administered to treat cutaneous leishmaniasis is miltefosine, proven to be efficacious in clinical trials. Preventive measures include avoiding sandfly bites and applying insecticides in areas prone to L. major.
Essentiality of this protein: essential after 6 days Gene/Ortholog: Tb09.v1.0350 (OG4_15067); Phenotype: significant loss of fitness in bloodstream forms (6 days); Source study: alsford alsford: High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome. Genome Res 2011, 21:915-924 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3106324/ Trypanosoma brucei: protozoan and order = trypanosomatida Leishmania major: protozoan and order = trypanosomatida
Complex of proteins?: No
Druggable Target: Druggability index (range: 0 to 1): 0.2
Enzyme Assay information (spectrophotometric, coupled assay?, reagents): type of enzyme assay: spectrophotometric -- link to Sigma (or other company) page for assay or assay reagents (substrates): http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/ptp101bul.Par.0001.File.tmp/ptp101bul.pdf -- link (or citation) to paper that contains assay information: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3097125/ -- List cost and quantity of substrate reagents and supplier:
10 µl pNP. (5 mg tablets - $32.60, Sigma Aldrich)
250 mM 1× phosphatase buffer ($89.30, Sigma Aldrich)
1N NaOH ($18.50, Sigma Aldrich)
40 µl immunoprecipitated protein ($162.50, Sigma Aldrich)
Structure Available (PDB or Homology model)
-- PDB #: 3S4O
Expression Information (has it been expressed in bacterial cells):
Expressed in E. coli
Purification Method:
Chromatographic separation on fast flow Q-Sepharose, heparin-agarose, l-histidyldiazobenzylphosphonic acid agarose, and hydroxylapatite
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MGIKDMYLLAYNAGMCVGWGTILVKIIVHLAEGRDPASVYPDIARLLCVAQTGAVAEIFHAAFGIVRSPV GTTFLQVLSRLIVLYGAVRIGDTDSTKSLVFVQMLVAWCLSEIIRYSFYGANLLRVNVASLTWLRYSAFM VLYPVGITGEIGCLYKALPYIKKHKPWTVELPNKLNFTFSWYNAVWFILLGIYPYGSYVMYSYMLAQRRK TFAKAAPERSKKSA
*length of your protein in Amino Acids: 224 Amino Acids
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite: 25166.8 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength:
Ext. coefficient 57090 Abs 0.1% (=1 g/l) 2.268, assuming all pairs of Cys residues form cystines
Ext. coefficient 56840 Abs 0.1% (=1 g/l) 2.259, assuming all Cys residues are reduced
*CDS Gene Sequence (paste as text only): ATGGAGGTGAACGCTACGCTGATCGACTGCTGCGACCCGCAGAAGCCGTCGAGGGTGCTCTTTCACTTTC TCATCCTCGACGCGCCGTCCCCCAGCAACCTCCCGACATACATCAAGGAGCTGCAGCACCGCGGCGTGCG GCACTTGGTGCGGGTATGCGGCCCGACCTACGACGCCACCCTTGTCAAGAGCAGGGGCATTGATGTCCAC AGCTGGCCATTCGACGACGGCGCGCCTCCGACGCGTGCGGTGCTTGACAGCTGGCTGAAGCTGCTCGACA CGGAGCTTGCGCGTCAGCAGGAGGATCCCTCCGTGCCTCCGCCAACCATCGGCGTGCACTGCGTGGCGGG CCTGGGCCGCGCACCAATCTTGGTGGCGCTGGCGCTGGTGGAGTACGGCAACGTATCTGCACTAGACGCT ATCGCACTGATCCGCGAGAAGCGCAAGGGTGCAATTAATCAGACACAGATGCACTGGATCACCAAGTACA AGCGTCGCCACCAGGGTGCGGGCTGCGTGATCATGTAG
*GC% Content for gene: 33.9%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized): Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
*NCBI Gene # or RefSeq#:
Gene symbol: LMJF_05_0280
Gene ID: 5648889
*Protein ID (NP or XP #) or Wolbachia#:
TriTrypDB / LmjF04.0560 GeneDB / LmjF04.0560
*Organism (including strain): Leishmania major strain Friedlin
Etiologic Risk Group (see link below): Risk group 2 (RG2) - parasitic agents
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Leishmania major is a protozoan species associated with the disease zoonotic cutaneous leishmaniasis (the most common form of leishmaniasis). The intracellular pathogen affects hosts' immune systems by infecting dendritic cells and macrophages. L. major is largely endemic to the Eastern hemisphere, specifically the Middle East, and the northern regions of Africa, China, and India. However, leishmaniasis is also found in the Americas, and has even recently spread to North Texas. There are approximately 1-1.5 million worldwide cases of cutaneous leishmaniasis a year. The disease is transmitted by the bite of female sandflies (spread by vectors). Clinical presentations of cutaneous leishmaniasis include acute infection and lesions at the bite site. Nodules that ulcerate may form. Sores may remain for a few months to a year. The patient's immune system is usually naturally restored within 3-6 months after infection. A current antiprotozoal oral drug administered to treat cutaneous leishmaniasis is miltefosine, proven to be efficacious in clinical trials. Preventive measures include avoiding sandfly bites and applying insecticides in areas prone to L. major.
Essentiality of this protein: essential after 6 days
Gene/Ortholog: Tb09.v1.0350 (OG4_15067); Phenotype: significant loss of fitness in bloodstream forms (6 days); Source study: alsford
alsford: High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome. Genome Res 2011, 21:915-924
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3106324/
Trypanosoma brucei: protozoan and order = trypanosomatida
Leishmania major: protozoan and order = trypanosomatida
Complex of proteins?: No
Druggable Target: Druggability index (range: 0 to 1): 0.2
*EC#: 3.1.3.48
Link to BRENDA EC# page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.48&Suchword=&organism%5B%5D=Leishmania+major+Friedlin+V9&show_tm=0
-- Show screenshot of BRENDA enzyme mechanism schematic:
Enzyme Assay information (spectrophotometric, coupled assay?, reagents): type of enzyme assay: spectrophotometric
-- link to Sigma (or other company) page for assay or assay reagents (substrates):
http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/ptp101bul.Par.0001.File.tmp/ptp101bul.pdf
-- link (or citation) to paper that contains assay information:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3097125/
-- List cost and quantity of substrate reagents and supplier:
10 µl pNP. (5 mg tablets - $32.60, Sigma Aldrich)
250 mM 1× phosphatase buffer ($89.30, Sigma Aldrich)
1N NaOH ($18.50, Sigma Aldrich)
40 µl immunoprecipitated protein ($162.50, Sigma Aldrich)
Structure Available (PDB or Homology model)
-- PDB #: 3S4O
Current Inhibitors:
alendronate
galectin-1 (for CD45)
4-Isoavenaciolide
tetronic acid derivative RK-682
Expression Information (has it been expressed in bacterial cells):
Expressed in E. coli
Purification Method:
Chromatographic separation on fast flow Q-Sepharose, heparin-agarose, l-histidyldiazobenzylphosphonic acid agarose, and hydroxylapatite
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MGIKDMYLLAYNAGMCVGWGTILVKIIVHLAEGRDPASVYPDIARLLCVAQTGAVAEIFHAAFGIVRSPV GTTFLQVLSRLIVLYGAVRIGDTDSTKSLVFVQMLVAWCLSEIIRYSFYGANLLRVNVASLTWLRYSAFM VLYPVGITGEIGCLYKALPYIKKHKPWTVELPNKLNFTFSWYNAVWFILLGIYPYGSYVMYSYMLAQRRK TFAKAAPERSKKSA
*length of your protein in Amino Acids: 224 Amino Acids
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite: 25166.8 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength:
Ext. coefficient 57090 Abs 0.1% (=1 g/l) 2.268, assuming all pairs of Cys residues form cystines
Ext. coefficient 56840 Abs 0.1% (=1 g/l) 2.259, assuming all Cys residues are reduced
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
ATGGAGGTGAACGCTACGCTGATCGACTGCTGCGACCCGCAGAAGCCGTCGAGGGTGCTCTTTCACTTTC TCATCCTCGACGCGCCGTCCCCCAGCAACCTCCCGACATACATCAAGGAGCTGCAGCACCGCGGCGTGCG GCACTTGGTGCGGGTATGCGGCCCGACCTACGACGCCACCCTTGTCAAGAGCAGGGGCATTGATGTCCAC AGCTGGCCATTCGACGACGGCGCGCCTCCGACGCGTGCGGTGCTTGACAGCTGGCTGAAGCTGCTCGACA CGGAGCTTGCGCGTCAGCAGGAGGATCCCTCCGTGCCTCCGCCAACCATCGGCGTGCACTGCGTGGCGGG CCTGGGCCGCGCACCAATCTTGGTGGCGCTGGCGCTGGTGGAGTACGGCAACGTATCTGCACTAGACGCT ATCGCACTGATCCGCGAGAAGCGCAAGGGTGCAATTAATCAGACACAGATGCACTGGATCACCAAGTACA AGCGTCGCCACCAGGGTGCGGGCTGCGTGATCATGTAG
*GC% Content for gene: 33.9%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
References:
http://www.metalife.com/PDB/3S4O_76577
http://biocyc.org/LEISH/NEW-IMAGE?type=REACTION&object=PROTEIN-TYROSINE-PHOSPHATASE-RXN
http://tdrtargets.org/targets/view?gene_id=26652
http://www.sigmaaldrich.com/catalog/search?interface=All&term=protein%20tyrosine%20phosphatase%20&lang=en®ion=US&focus=product&N=0+220003048+219853269+219853286
http://www.rcsb.org/pdb/explore/explore.do?structureId=3S4O
http://www.genomicsplace.com/gc_calc.html
http://tritrypdb.org/tritrypdb/showRecord.do?name=GeneRecordClasses.GeneRecordClass&primary_key=LmjF16.0230
http://onlinelibrary.wiley.com/doi/10.1002/anie.200461517/full
http://www.ncbi.nlm.nih.gov/pubmed/12112018
http://www.ncbi.nlm.nih.gov/gene/5648889